Germinated broad bean feed for improving intestinal health of tilapia mossambica and preparation method thereof

Abstract

The invention belongs to the technical field of feed processing, and particularly relates to germinated broad bean feed for improving intestinal health of tilapia mossambica and a preparation method of the germinated broad bean feed. The germinated broad bean feed capable of improving intestinal health of tilapia mossambica provided by the invention is mainly prepared from the following raw materials: fish meal, soybean meal, cottonseed meal, rapeseed meal, wheat bran, rice bran, wheat middling, soybean oil, choline chloride, a vitamin premix, a mineral element premix, monocalcium phosphate, germinated broad beans, a mildew preventive and the like. The germinated broad bean feed prepared by the invention not only can reduce the diameter of muscle fibers of tilapia mossambica muscle and increase the density, but also can increase the hardness and collagen content of the muscle, so that the meat quality of the tilapia mossambica is more compact, tasty and crisp, and the breeding quality is improved. Meanwhile, the germinated broad bean feed can also improve intestinal inflammation of the tilapia mossambica, promote intestinal health, improve the immune defense function of the tilapia mossambica and improve the health condition of the crisp tilapia mossambica, and can effectively promote sustainable development of the crisp tilapia mossambica industry.

Description

Technical Field

[0001] The present invention belongs to the technical field of feed processing, and particularly relates to a germinated broad bean feed for improving the intestinal health of tilapia and a preparation method thereof. Background Art

[0002] Nile tilapia (Oreochromis niloticus), belonging to the family Cichlidae of the order Perciformes, is a tropical fish widely cultured globally. This fish species is highly favored for its rapid growth, strong environmental adaptability, excellent meat quality, and convenience in aquaculture management. Despite the wide recognition of its economic and nutritional value, the muscle quality of Nile tilapia still faces many challenges, mainly manifested as problems such as soft meat quality, poor taste, and obvious earthy smell in the freshwater pond culture mode, which severely limit its edible value and export potential.

[0003] Research shows that feeding broad beans can significantly improve the meat quality of various fish such as tilapia. However, there are many problems with directly feeding broad beans, such as poor palatability and slow growth. Especially under the special digestive system conditions of Nile tilapia, it may lead to high mortality. In addition, although the special brittle feed pellets are effective to a certain extent, their high feed conversion ratio and potential risks such as hepatobiliary diseases limit their wide application. In this context, searching for new feed additives has gradually been applied to aquaculture and has received increasing attention.

[0004] Patent document CN113729128A discloses a feed additive for promoting the crispness of tilapia meat. The feed additive is mainly composed of raw materials such as broad bean water extract and amino acid complex. The feed additive can significantly improve the degree of crispness of tilapia muscle, increase the muscle fiber diameter, increase the muscle hardness, and enhance the chewability. Compared with feeding pure broad beans, the growth rate of tilapia is faster.

[0005] Patent document CN117694470A discloses a brittle artificial compound feed for tilapia and a preparation method thereof. The feed is mainly composed of raw materials such as broad beans, flour, fish meal, oil crop meal, purple flower beans, spinach powder, triterpenoid saponin extract, compound vitamin premix, compound trace element premix, compound enzyme, binder, etc. The feed makes the tilapia meat brittle by adding broad beans, adds spinach powder and purple flower beans to increase the content of methionine, cysteine, and tryptophan to support the growth of tilapia, adds compound vitamins and compound organic trace elements to meet the growth needs of tilapia. Adding compound enzymes promotes the digestion and absorption of the feed in tilapia and accelerates the growth of tilapia. In addition, adding triterpenoid saponin extract binds to the fat in the tilapia intestine, promotes the digestion and absorption of fat, and reduces the accumulation of fat in the intestine, thereby reducing the fishy smell and muddy smell.

[0006] However, due to the large amount of anti-nutritional substances in broad beans, such as trypsin inhibitors and condensed tannins, long-term ingestion of broad beans or broad bean powder feed can cause inflammation in the intestines of crispy tilapia, slow growth rate, poor crisping effect, and death, which greatly affects the development of the crispy tilapia industry. Summary of the Invention

[0007] In order to solve the defects existing in the prior art, the present invention provides a germinated broad bean feed for improving the intestinal health of tilapia and a preparation method thereof. The present invention creatively uses germinated broad beans as an additive to the crispy tilapia feed to solve the problems of unbalanced nutrient elements in broad beans and the obstruction of the absorption of nutrients by tilapia due to endogenous anti-nutritional factors. The feed prepared from germinated broad beans for a specific number of days can not only improve the growth performance and immune ability of tilapia, but also maintain the intestinal health of tilapia, and can effectively promote the development of the crispy tilapia industry.

[0008] The specific technical solutions are as follows:

[0009] The present invention provides a germinated broad bean feed for improving the intestinal health of tilapia, comprising the following components and their weight parts:

[0010] Fish meal 1 - 3 parts, soybean meal 10 - 20 parts, cottonseed meal 5 - 20 parts, rapeseed meal 5 - 20 parts, wheat bran 8 - 12 parts, rice bran 8 - 12 parts, wheat middlings 10 - 25 parts, soybean oil 1 - 2 parts, choline chloride 0.4 - 0.6 parts, vitamin premix 0.2 - 0.4 parts, mineral element premix 0.2 - 0.5 parts, calcium dihydrogen phosphate 1 - 3 parts, germinated broad beans 45 - 55 parts, and mold inhibitor 0.1 - 0.2 parts.

[0011] Further, the germinated broad bean feed for improving the intestinal health of tilapia consists of the following components and their weight parts:

[0012] Fish meal 1 part, soybean meal 10 parts, cottonseed meal 6 parts, rapeseed meal 8 parts, wheat bran 10 parts, rice bran 10 parts, wheat middlings 10.85 parts, soybean oil 1 part, choline chloride 0.5 part, vitamin premix 0.25 part, mineral element premix 0.3 part, calcium dihydrogen phosphate 1.5 parts, germinated broad beans 50 parts, and mold inhibitor 0.15 part, and the mold inhibitor is montmorillonite.

[0013] Further, the germinated broad beans are the germinated broad beans on the 1st - 5th day after the broad beans are soaked and germinated.

[0014] Further, the mold inhibitor is modified montmorillonite.

[0015] Further, the preparation method of the modified montmorillonite is:

[0016] Step A: Add calcium-based montmorillonite into distilled water, and hydrate for 1 - 2 h to obtain a suspension; then add sodium carbonate into the suspension, react at a temperature of 50 - 70 °C for 1 - 3 h, dry, and crush to 1000 - 1500 meshes to obtain modified sodium-based montmorillonite;

[0017] Step B: Add arginine and the modified sodium-based montmorillonite prepared in Step A into distilled water, stir and react for 1 - 3 h, centrifuge, wash the precipitate until it is neutral, and dry in vacuum to obtain the product.

[0018] Furthermore, in Step A, the material-liquid ratio of calcium-based montmorillonite to distilled water is 1 g : (8 - 10) mL.

[0019] Furthermore, in Step A, the mass concentration of sodium carbonate in the suspension is 4 - 5%.

[0020] Furthermore, in Step B, the mass ratio of arginine to modified sodium-based montmorillonite is (5 - 7) : 10.

[0021] Furthermore, in Step B, the material-liquid ratio of modified sodium-based montmorillonite to distilled water is 1 g : (6 - 8) mL.

[0022] In addition, the present invention also provides a preparation method of the germinated broad bean feed for improving the intestinal health of tilapia, comprising the following steps:

[0023] Step S1: Mix fish meal, soybean meal, cottonseed meal, rapeseed meal, wheat bran, rice bran, wheat middlings, choline chloride, vitamin premix, mineral element premix, calcium dihydrogen phosphate, germinated broad beans and mold inhibitor evenly, crush, and pass through a 40-mesh sieve to obtain mixture I;

[0024] Step S2: Add soybean oil and distilled water into mixture I prepared in Step S1, the addition amount of the distilled water is 25 - 30% of the mass of mixture I, stir evenly, make into sinking particles with a particle size of 2 mm, and dry at a temperature of 60 - 70 °C to obtain the product.

[0025] The present invention studies the effects of feeds made from broad beans with different germination days on the growth performance of tilapia and the crispness of fish meat. Through experiments, it is found that germination can reduce the anti-nutritional factors of broad beans and improve the nutritional value of broad beans. At the same time, the germinated broad bean feed provided by the present invention can make the muscle fiber diameter of tilapia thinner and the density increase, increase the muscle hardness and collagen content, so that the meat quality of tilapia is more firm and crispy, improving the aquaculture quality. In addition, the inventors also found that the germinated broad bean feed has a positive impact on the growth and development, antioxidant system and intestinal health of tilapia. Specifically, the germinated broad bean feed can improve the growth performance of tilapia, reduce the oxidative stress of tilapia body and reduce the incidence of intestinal inflammation in tilapia, can effectively improve the immune defense function of crisp tilapia, improve the health status of crisp tilapia, and can promote the sustainable development of the crisp tilapia industry.

[0026] In addition, during the research process, the inventors found that there are generally mycotoxin contaminants in aquatic feeds made by replacing animal protein sources with plant protein sources. Deoxynivalenol (DON) is a toxic secondary metabolite produced by Fusarium moniliforme, and its detection rate and exceeding standard rate are extremely high in aquatic feeds with plant protein sources, seriously endangering the food safety of crisp tilapia.

[0027] Therefore, the inventors added montmorillonite as a mold inhibitor to the germinated broad bean feed. Through research, it is found that montmorillonite has good adsorption ability for DON and can remove most of the DON in the feed. However, the feed is prone to nourish molds during storage, and relying solely on the physical adsorption of montmorillonite cannot achieve a good effect of removing DON. After a large number of exploratory experiments, the inventors prepared a modified montmorillonite to further reduce the content of DON. The present invention uses arginine to modify montmorillonite, so that arginine is inserted into the interlayer of montmorillonite, making the interlayer spacing of montmorillonite larger, thereby improving the adsorption ability of montmorillonite for DON, and the arginine loaded in montmorillonite can also inhibit the biosynthesis of deoxynivalenol, reducing the content of deoxynivalenol from the source.

[0028] In addition, the inventors also found that the modified montmorillonite can also adsorb the anti-nutritional component tannin in broad bean powder in the tilapia body, reducing the impact of tannin on tilapia. At the same time, the modified montmorillonite and the germinated broad beans can synergistically improve the growth performance of crisp tilapia, effectively relieve the occurrence of symptoms such as loss of appetite, weight loss, and metabolic disorders caused by components such as tannin or DON in tilapia, and improve the health status of crisp tilapia.

[0029] In summary, compared with the prior art, the germinated broad bean feed for improving the intestinal health of tilapia provided by the present invention can not only make the muscle fiber diameter of tilapia thinner and the density increase, but also increase the muscle hardness and collagen content, thereby making the meat quality of tilapia more firm and crispy, and improving the aquaculture quality. At the same time, the germinated broad bean feed can also improve the intestinal inflammation of tilapia, promote intestinal health, enhance the immune defense function of tilapia, improve the health status of crispy tilapia, and effectively promote the sustainable development of the crispy tilapia industry. Description of the Drawings

[0030] Figure 1 It is a graph showing the results of the analysis of the muscle texture of tilapia fed with germinated broad bean feed for different days.

[0031] Figure 2 It is a graph showing the results of the change in the muscle collagen content of tilapia fed with germinated broad bean feed for different days.

[0032] Figure 3 It is a microstructural diagram of the muscle of tilapia fed with germinated broad bean feed for different days.

[0033] Figure 4 It is a histological structure diagram of the liver of tilapia fed with germinated broad bean feed for different days.

[0034] Figure 5 It is a graph showing the expression results of non-specific immune-related genes in the intestine of tilapia fed with germinated broad bean feed for different days.

[0035] Figure 6 It is an observation diagram of the microstructure of the intestine of tilapia fed with germinated broad bean feed for different days. Detailed Embodiments

[0036] The present invention will be further described below through the description of specific embodiments. However, this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of the present invention, but as long as they do not deviate from the basic idea of the present invention, they are within the scope of the present invention. The materials and reagents involved in the present invention are all food-grade and can be obtained through commercial purchase or conventional technical means in the art.

[0037] I. Preparation of a germinated broad bean feed for improving the intestinal health of tilapia

[0038] 1. The formula of the germinated broad bean feed for improving the intestinal health of tilapia is shown in Table 1:

[0039] Table 1 Composition and nutrient content of germinated broad bean feed (air-dried weight, g / kg)

[0040]

[0041]

[0042] Note: FB0 broad beans are dry broad beans without germination, FB1 broad beans are broad beans germinated for 1 day, and FB5 broad beans are broad beans germinated for 5 days.

[0043] 2. Preparation method of germinated broad beans:

[0044] Before germination, the broad beans are washed 4 times with sterile water to remove possible bacterial contaminants. The washed beans are placed on wet filter paper in a plate and stored in an incubator for 1 day and 5 days; a small amount of water is added to the plate regularly to keep it moist. Seeds that do not germinate during this period are discarded. After germination, the broad beans of 1 day and 5 days are collected, freeze-dried, and ground into small particles.

[0045] 2. Preparation method of germinated broad bean feed:

[0046] Step S1: Mix fish meal, soybean meal, cottonseed meal, rapeseed meal, wheat bran, rice bran, wheat middlings, choline chloride, vitamin premix, mineral element premix, calcium dihydrogen phosphate, germinated broad beans and montmorillonite evenly, crush them, and pass through a 40-mesh sieve to obtain mixture I;

[0047] Step S2: Add soybean oil and distilled water to the mixture I prepared in Step S1. The addition amount of the distilled water is 30% of the mass of the mixture I. Stir evenly, make sinking particles with a particle size of 2 mm, and dry them at a temperature of 65 °C to obtain the product.

[0048] Example 3. Preparation method of modified montmorillonite

[0049] Step A: Add calcium-based montmorillonite to distilled water. The material-liquid ratio of the calcium-based montmorillonite to distilled water is 1 g:8 mL, and hydrate for 2 h to obtain a suspension; then add sodium carbonate to the suspension. The mass concentration of the sodium carbonate in the suspension is 5%, and react at a temperature of 60 °C for 2 h, dry, and crush to 1000 - 1500 meshes to obtain modified sodium-based montmorillonite;

[0050] Step B: Add arginine and the modified sodium-based montmorillonite prepared in Step A to distilled water. The material-liquid ratio of the modified sodium-based montmorillonite to distilled water is 1 g:6 mL, and the mass ratio of the arginine to the modified sodium-based montmorillonite is 7:10. Stir and react for 2 h, centrifuge, wash the precipitate until neutral, and vacuum dry to obtain the product.

[0051] Example 4. Preparation of a germinated broad bean feed for improving the intestinal health of tilapia

[0052] The difference from Example 2 is that montmorillonite is replaced with the modified montmorillonite prepared in Example 3, and the other components and steps are similar to those in Example 2.

[0053] Example 5: Collection and Processing of Nile Tilapia Samples

[0054] 1. Experimental Grouping:

[0055] The tilapia used in the experiment was purchased from Zhaoqing Fisheries Science Research Institute and acclimated in the basal diet (the diet of Control Example 1) for 2 weeks before the experiment. Tilapia with strong physique and uniform specifications were selected for the experiment, with an average body weight of 250 ± 10.0 g. 360 tilapia were randomly assigned to 12 cages, with 3 replicates in each experimental group and 30 fish in each cage.

[0056] During the experiment, feeding was carried out 2 times a day (at 9:00 am and 5:00 pm), and the daily feeding rate was 3.0% of the fish body weight, adjusted according to water temperature and feeding situation. The feeding amount in each cage was kept basically the same. The death situation was checked daily during the breeding period, and the food intake and the number of deaths were recorded. The water temperature was kept at about 28 °C during the breeding period. The sewage was sucked and the water was changed twice a week, and the water quality indicators were measured regularly every day. The water quality conditions included dissolved oxygen > 5 mg / L, water temperature 25 - 30 °C, pH 7.5 - 8.0, ammonia nitrogen < 0.2 mg / L, and nitrite < 0.1 mg / L. The breeding cycle was 80 days. Broad beans with different germination days were added to the basal diet, as shown in Table 1 specifically. The groups were named Control group (Control Example 1), FB0 group (Control Example 2), FB1 group (Example 1), and FB5 group (Example 2).

[0057] 2. Experimental Samples:

[0058] Samples were collected once at 15 d, 45 d, and 80 d of the breeding time. The day before sampling, the fish were starved for 24 h. The number of grass carp in each cage was counted and weighed. 3 tilapia were randomly selected from each cage to measure body weight, body length, etc., and then blood was collected from the caudal vein to obtain serum, which was stored in a -80 °C refrigerator for subsequent experiments. After blood collection, the tilapia were immediately dissected, and the visceral weight and liver weight were weighed. The liver, intestine, intestinal contents of the fish, and muscle samples collected from the left side of the tilapia were frozen and stored at -80 °C for subsequent experimental analysis; 1 piece of liver, intestine, and muscle were respectively stored in the fixing solution for histological section observation; another 3 pieces of dorsal muscle about 1 cm 3 in size were taken from the right side for subsequent texture analysis.

[0059] Experimental Example 1: Effect of Germinated Broad Bean Feed on the Muscle Texture of Tilapia

[0060] 1. Experimental Method:

[0061] To investigate the effect of germinated broad beans added to the feed for different days on the texture of tilapia muscle, the specific grouping is shown in Example 5. The breeding cycle is 80 days. Samples are taken at 15d, 45d, and 80d. The texture characteristics and muscle collagen content of tilapia in each group are detected, and the microscopic structure of the muscle is observed.

[0062] 1.1 Determination of muscle texture:

[0063] A Universal TA type texture analyzer equipped with a P35 cylindrical probe is used to evaluate the texture characteristics of fish meat, including hardness, elasticity, chewiness, cohesiveness, and resilience. The parameter settings for operating this test are as follows: the speed before the indenter moves to the surface of the sample is 1.00 mm / s, the speed during the compression test is 1.00 mm / s, the speed after the indenter returns to the original position is also 1.00 mm / s, the compression ratio is 25%, and the measurement interval time between two measurements is 2 s; the trigger force is 8 g. The ambient temperature is controlled at 20 - 25 °C. Each sample is measured 4 times, and the average value is taken.

[0064] 1.2 Determination of muscle collagen content:

[0065] With reference to AOAC (2000), the hydroxyproline content of muscle samples is first determined. The determination of hydroxyproline content uses the alkali hydrolysis method and a kit provided by Nanjing Jiancheng Biotechnology Research Institute. The collagen content is obtained by multiplying the hydroxyproline content by 8 according to the AOAC method.

[0066] 1.3 Observation of the microscopic structure of muscle:

[0067] The muscle samples are sectioned, and the sections are stained with hematoxylin - eosin dye. The clear structures of cross - sectioned and longitudinally - sectioned muscle fibers are observed under an optical microscope.

[0068] 2. Experimental results:

[0069] The experimental results are as Figures 1 to 3 shown.

[0070] 2.1 The determination results of tilapia muscle texture are as Figure 1 shown:

[0071] Figure 1 It is a graph of the effect of feeding tilapia with germinated broad bean feed for different days on muscle texture analysis, where: Figure 1 - a is hardness, Figure 1 - b is chewiness, Figure 1 - c is elasticity.

[0072] From Figure 1 it can be seen that:

[0073] (1) Determination of the muscle hardness of tilapia: At 15 d, the hardness of the Control group was 89.03 ± 7.60 gf, that of the FB0 group was 128.09 ± 9.41 gf, that of the FB1 group was 147.88 ± 11.41 gf, and that of the FB5 group was 157.93 ± 11.04 gf. At 45 d, the hardness of the Control group was 145.17 ± 21.66 gf, while that of the FB0 group was 238.11 ± 20.80 gf, that of the FB1 group was 305.04 ± 34.99 gf, and that of the FB5 group was 389.33 ± 51.63 gf (P < 0.05). As the breeding days increased, the hardness of tilapia gradually increased. When the breeding days were 80 d, the hardness of the FB5 group was the highest, which was 168.19% higher than that of the Control group, 63.50% higher than that of the FB0 group, and 29.18% higher than that of the FB1 group.

[0074] (2) Determination of the chewiness of tilapia muscle: The chewiness of the Control group, FB0 group, FB1 group, and FB5 group gradually increased at 15 d, 45 d, and 80 d. When the breeding days were 80 d, the chewiness of the Control group, FB0 group, FB1 group, and FB5 group was 55.02 ± 8.86 gf, 104.77 ± 11.91 gf, 143.79 ± 16.53 gf, and 168.00 ± 35.30 gf respectively (P < 0.05).

[0075] (3) Determination of the elasticity of tilapia muscle: The elasticity of the FB5 group was significantly higher than that of the Control group, FB0 group, and FB1 group (P < 0.05).

[0076] The above results show that the muscle hardness and chewing force of tilapia fed with germinated broad bean feed for different days were significantly higher than those of the Control group at 15 d, 45 d, and 80 d (P < 0.05). Among them, at 80 d, the muscle hardness of the FB5 group was the highest, which was 168.19%, 63.50%, and 29.18% higher than that of the Control group, FB0 group, and FB1 group respectively. It is proved that the germinated broad bean feed provided by the present invention can improve the texture characteristics of tilapia muscle, and the broad beans germinated for 5 days in the FB5 group have the best effect.

[0077] 2.2. The determination results of the collagen content in tilapia muscle are as Figure 2 shown:

[0078] Figure 2 It is a result graph of the change in the collagen content in tilapia muscle fed with germinated broad bean feed for different days. From Figure 2It can be seen that with the increase of the breeding days, the content of collagen in each group gradually increases. At 15 d, the muscle collagen content in the FB0 group is the highest. At 45 d, the muscle collagen contents in the FB0 group, FB1 group and FB5 group are higher than those in the Control group. At 80 d, the muscle collagen contents in the FB0 group, FB1 group and FB5 group are higher than those in the Control group, and the collagen contents in the FB1 and FB5 groups are higher than those in the FB0 group. It shows that germinated broad beans are helpful to increase the muscle collagen content.

[0079] 2.3 The microscopic structure observation results of tilapia muscle are as Figure 3 shown:

[0080] Figure 3 It is the microscopic structure diagram (×20 times) of tilapia muscle fed with germinated broad bean feed for different days.

[0081] From Figure 3 it can be known that:

[0082] (1) According to the observation of the muscle cross-section microscopic structure diagram at 20 times magnification, compared with the Control group, with the extension of the crisping time, the muscle fiber diameters of tilapia in the FB0 group, FB1 group and FB5 group gradually decrease, and the number of muscle fibers in the same field of view gradually increases, and the FB5 group at 80 d of crisping shows the most significant performance. At the same time, the number of muscle fibers in the same area also increases with the extension of the feeding time, and the number of muscle fibers per square millimeter is the highest in the FB5 group at 80 d of crisping.

[0083] (2) According to the further display of the muscle longitudinal-section microscopic structure diagram, with the increase of the crisping time, the muscle fiber diameters of tilapia in the FB0 group, FB1 group and FB5 group gradually decrease, the number of muscle fibers in the same field of view gradually increases, and further decreases with the increase of the feeding time of germinated broad beans. In the FB5 group at 80 d of crisping, the muscle fiber diameter is the smallest.

[0084] The above results show that compared with the FB0 group and FB5 group, the cross-sectional area of tilapia muscle fibers in the FB5 group is smaller, the longitudinal-section muscle fiber diameter is less, and the number of muscle fibers in the same area increases more.

[0085] Experimental Example 2: Effects of germinated broad bean feed on the growth performance of tilapia

[0086] 1. Experimental method:

[0087] In order to explore the effects of adding germinated broad beans with different days to the feed on the growth performance of tilapia, the specific grouping is as shown in Example 5. The breeding cycle is 80 days. Samples are taken at 15 d, 45 d and 80 d, and the growth indexes and physical indexes of tilapia in each group are detected. Specifically:

[0088] Survival rate (survival, %) = 100 × Nt / No;

[0089] Weight gain rate (weight gain, WG, %) = 100 × (Wt - Wo) / Wo;

[0090] Hepatosomatic index (HSI, %) = 100 × Wh / W;

[0091] Viscerosomatic index (VSI, %) = 100 × Wv / W;

[0092] Condition factor (CF, g / cm 3 ) = 100 × W / L 3 ;

[0093] Where, Nt is the final number; No is the initial number; Wt is the final body weight (g); Wo is the initial body weight (g); Wh is the liver weight of the fish (g); Wv is the visceral weight of the fish (g); W is the body weight of the fish (g); L is the body length of the fish (cm).

[0094] 2. Experimental results:

[0095] The experimental results are shown in Table 2.

[0096] Table 2 Effects of diets of germinated broad beans fed for different days on the growth performance of tilapia

[0097]

[0098]

[0099] As can be seen from Table 2:

[0100] (1) At 15 d of cultivation, the survival rate of tilapia in the FB0 group during the cultivation period was 98%, and the survival rates of the Control group, FB1 group and FB5 group during the cultivation period were all 100%. The visceral index and hepatosomatic index (HSI) were significantly lower than those of the control group (P < 0.05). There were no significant differences in the weight gain rate, body length and condition factor between the experimental groups and the control group (P > 0.05).

[0101] (2) At 45 days of cultivation, the survival rates of tilapia in the FB0 group, FB1 group, and FB5 group were all 100% during the cultivation period, the same as that of the tilapia in the Control group. The weight gain rate of tilapia in the FB0 group was significantly lower than that of the control group (P>0.05), while there was no significant difference in the weight gain rate of tilapia in the FB1 group and FB5 group compared with the control group (P<0.05). There was no significant difference in body length, visceral index, and hepatosomatic index of tilapia in the FB0 group, FB1 group, and FB5 group compared with those in the Control group (P>0.05). The condition factor of tilapia in the FB1 and FB5 groups had no significant difference compared with that of the control group (P>0.05), and the condition factor of tilapia in the FB5 group was significantly higher than that of the FB0 group (P>0.05).

[0102] (3) At 80 days of cultivation, after the cultivation experiment, the survival rates of tilapia in the FB0 group, FB1 group, and FB5 group were all 100% during the cultivation period, the same as that of the tilapia in the Control group. The weight gain rate of tilapia in the FB0 group was significantly lower than that of the control group (P>0.05), only 61.77%, while there was no significant difference in the weight gain rate of tilapia in the FB1 group and FB5 group compared with the Control group (P<0.05). The weight gain rate and condition factor of tilapia in the FB0 group were significantly lower than those of the control group (P>0.05), while there was no significant difference in the weight gain rate of tilapia in the FB1 group and FB5 group compared with the control group (P<0.05).

[0103] The above results show that compared with the FB0 group, the germinated broad bean feed provided by the present invention can improve the weight gain rate and condition factor of tilapia, especially the broad beans germinated for 5 days in the FB5 group have the best effect.

[0104] Experimental Example 3. Effects of germinated broad bean feed on non-specific immunity of tilapia liver and blood

[0105] 1. Experimental method:

[0106] In order to explore the effects of adding germinated broad beans with different days to the feed on the growth performance of tilapia, the specific grouping is shown in Example 5. The cultivation period is 80 days. Samples are taken at 15d, 45d, and 80d, and the antioxidant enzyme activity indexes, serum and liver injury enzyme activity indexes, and serum and liver non-specific indexes of tilapia in each group are detected, and the microscopic structure of the liver is observed.

[0107] 1.1 Determination of enzyme activity indexes:

[0108] (1) Preparation of tissue homogenate: Serum samples and liver samples previously stored frozen at -80°C were taken out. Subsequently, six tilapia were selected from each of the total 4 groups of tilapia, and 0.1 g of liver tissue was accurately weighed and placed into a clean EP tube. 1 mL of protein extraction solution and 2 grinding beads were added, and the mixture was ground in a grinder at 4°C and 70 Hz for 2 min. When there were no obvious lumps after grinding, the mixture was centrifuged at 4°C and 5000 rpm for 15 min. After centrifugation, the floating fat on the upper layer was removed with a sterilized cotton swab, and then the supernatant was aspirated into a new EP tube and stored at 4°C.

[0109] (2) Determination of tissue homogenate: The experimental measurement of enzyme activities included the measurement of superoxide dismutase (SOD), reduced glutathione (GSH), catalase (CAT), malondialdehyde (MDA), glucose-6-phosphate dehydrogenase (G6PDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), acid phosphatase (ACP), and alkaline phosphatase (AKP). All enzyme activity detection work was completed according to the kit instructions provided by Nanjing Jiancheng Bioengineering Co., Ltd. The protein concentration of the corresponding samples was detected using an ultra-micro spectrophotometer.

[0110] 1.2. Determination of non-specific liver indicators:

[0111] 1.2.1. Total RNA extraction:

[0112] (1) Intestinal and liver samples previously refrigerated at -80°C were taken out of the refrigerator and slowly thawed on ice. Subsequently, approximately 50 mg of liver tissue was cut and placed into 1 mL of Trizol, and 100 μL of whole blood was placed into 900 μL of Trizol and 2 grinding beads. The mixture was ground in a grinder at 4°C and 70 Hz for 2 min until the tissue and whole blood were completely broken up and there were no obvious lumps. Subsequently, 200 μL of chloroform was added to each tube containing the sample tissue, and the mixture was vortexed for 30 s and then allowed to stand on ice for 10 min. The mixture was then centrifuged at 4°C and 12,000 rmp / min for 15 min.

[0113] (2) After centrifugation was completed, the EP tube was opened, and 400 μL of the upper clear liquid was gently transferred to a new centrifuge tube, and an equal volume of isopropanol was added. After gently inverting the tube 3 to 4 times to mix, the sample was allowed to stand at room temperature for 15 minutes and then centrifuged at 4°C and 12,000 revolutions per minute for 10 min.

[0114] (3) After centrifugation was completed, the supernatant was discarded, and 1 mL of 75% ethanol was carefully added to the tube for washing, and then centrifuged at 4°C and 12,000 rmp for 8 min. This washing process was performed twice to remove possible foreign substances.

[0115] (4) After centrifugation again, discard the supernatant, and use a small centrifuge to centrifuge the residual alcohol on the tube wall to the bottom of the tube. Gently aspirate the alcohol at the bottom using a pipette, and place the EP tube in a fume hood for 5 min to air-dry the residual alcohol, then add 20 to 40 μL of DEPC water to dissolve the RNA.

[0116] (5) Let the dissolution solution in the EP tube stand on ice for 15 to 20 min. Subsequently, use a ultra-micro spectrophotometer and agarose gel electrophoresis to detect the concentration and purity of the obtained total RNA.

[0117] 1.2.2, qRT-PCR:

[0118] Use a ultra-micro spectrophotometer to test the OD 260 / 280 value and the total RNA concentration. Then use the PrimeScript RT SYBR and Premix Ex Taq kits for reverse transcription and RT-qPCR assays. Search for and download the tilapia immune-related gene sequences according to the NCBI database, design the upstream and downstream primers for the tilapia immune-related genes using a website, use PCR amplification to screen the target primers, and select β-actin as the internal reference gene (see Table 3). The genes selected in this experiment are: interleukin 1β (IL-1β), heat shock protein 70 (Hsp70), tumor necrosis factor-α (TNF-α), metallothionein (MT), CC chemokine ligand 3 (CCL3), interferon-γ (IFN-γ). Using cDNA as a template, perform fluorescence quantitative PCR (qRT-PCR). The qRT-PCR system (20 μL): 10 μL of SYBR enzyme, 0.4 μL of each upstream and downstream primer, 4 μL of cDNA template, and 5.2 μL of water. The qRT-PCR reaction program: 95 °C for 30 s, 95 °C for 10 s, 60 °C for 30 s, 40 cycles. Based on the threshold cycle (Ct) value, calculate the relative expression level of the gene according to the 2 -ΔΔCt -method.

[0119] Table 3 Primers for internal reference gene and target genes

[0120]

[0121] 1.3, Observation of liver microstructure:

[0122] Process the liver samples into sections, stain the sections with hematoxylin-eosin dye, and observe the histological structure of the liver under an optical microscope.

[0123] 3. Experimental results:

[0124] The experimental results are shown in Tables 4 to 6 and Figure 4 as follows.

[0125] 3.1 Detection results of antioxidant enzyme activities in tilapia serum and liver are shown in Table 4 as follows:

[0126] Table 4 Antioxidant indices in tilapia liver and serum

[0127]

[0128]

[0129] It can be seen from Table 4 that:

[0130] (1) In tilapia liver: Compared with the Control group, the activities of MDA and SOD in the FB0 group and FB1 group were significantly enhanced within 15, 45, and 80 days, the GSH activity decreased significantly (P<0.05), but the CAT activities in the FB0 group and FB1 group were significantly enhanced within 15 and 45 days (P<0.05), but at 80 days, there was no significant difference in the CAT activity of the FB0 group (P>0.05), while that of the FB1 group was significantly enhanced (P<0.05). Meanwhile, the activities of MDA and SOD in the FB5 group were significantly enhanced within 15 and 45 days (P<0.05). The SOD level in the FB5 group was significantly increased (P<0.05), and there was no significant difference in the MDA level at 80 days (P>0.05). In addition, there was no significant difference in the CAT content in the liver of the FB5 group at 15 days (P>0.05), and the CAT activities in the FB5 group at 45 and 80 days were significantly higher than those in the control group. The GSH activity in the FB5 group decreased significantly at 15 days, but increased significantly at 45 and 80 days (P<0.05). Compared with the FB0 group, the MDA levels in the FB5 group decreased by 36.7%, 26.8%, and 44.2% respectively within 15, 45, and 80 days (P<0.05). Similarly, the SOD level in the FB5 group decreased significantly at 15 days (P>0.05), and decreased by 17.7% and 22.4% significantly at 45 and 80 days (P<0.05); the CAT activities decreased by 36.8% and 8.3% significantly at 15 and 45 days (P<0.05), but increased by 28.2% significantly at 80 days (P<0.05). In addition, the GSH activity in the FB5 group decreased by 45.8% significantly at 15 days, but increased by 105.4% and 65.9% significantly at 45 and 80 days (P<0.05). Compared with the Control group, the G6DPH content in the FB1 group was significantly up-regulated within 15, 45, and 80 days (P<0.05). There was no significant difference in the G6DPH activities between the Control group and the FB5 group within 15, 45, and 80 days (P>0.05). However, compared with the FB0 group, the G6DPH in the FB5 group at 15, 45, and 80 days (P<0.05).

[0131] (2) In tilapia serum: Compared with the Control group, the MDA content in the FB0 group, FB1 group, and FB5 group increased significantly within 15, 45, and 80 days (P<0.05). However, compared with the FB0 group, the MDA activity in the FB1 group and FB5 group decreased gradually by about 18.8% and 31.4%, respectively, and the changes at 15, 45, and 80 days were similar to those in the FB0 group. However, the CAT activity in the FB1 group and FB5 group was significantly lower than that in the Control group at 1 and 80 days, but significantly higher than that in the Control group at 45 days (P<0.05). Compared with the FB0 group, the CAT activity in the FB5 group decreased significantly at 15, 45, and 80 days (66.2%, 3.3%, and 74.5%, respectively). Compared with the Control group, the SOD activity in the FB0 group, FB1 group, and FB5 group increased significantly. At the same time, the SOD activity in the FB0 group was significantly lower at 45 and 80 days, but there was no significant difference in the FB1 group and FB5 group at 45 days, and the SOD activity in the FB1 group and FB5 group was significantly higher than that at 80 days. In addition, the GSH activity in the FB0 group, FB1 group, and FB5 group decreased significantly at 15 days. The GSH activity in the FB0 group and FB1 group increased significantly at 45 and 80 days, but there was no significant difference in the GSH activity in the FB5 group at 45 days compared with the Control group, and the GSH activity at 80 days decreased significantly. At different feeding times, the FB5 group was 3.0 times lower than the FB0 group. Compared with the Control group, the G6DPH content in the FB1 group increased significantly within 15 days, and the G6DPH activity in the FB1 group increased significantly within 45 days (P<0.05). Compared with the Control group, there was no significant difference in the G6DPH and its activity in the FB5 group (P>0.05). However, compared with the FB0 group, the G6DPH activity in the FB5 group decreased significantly by about 2.0 times within 15, 45, and 80 days (P<0.05).

[0132] 3.2. The detection results of the enzyme activities related to liver injury in tilapia serum are shown in Table 5 as follows:

[0133] Table 5 Enzyme activity indexes related to liver injury in serum

[0134]

[0135]

[0136] As can be seen from Table 5:

[0137] (1) In the liver of tilapia: Compared with the Control group, the activities of ACP, AKP, ALT, and AST in the FB0 group were significantly increased at 15, 45, and 80 days (P < 0.05). In the FB1 group, the activities of AKP, ACP, and ALT were significantly increased at 15, 45, and 80 days (P < 0.05), but the AST activity increased at 15, 45, and 80 days (P < 0.05). Compared with the Control group, the activities of AKP, ACP, and ALT in the FB1 group were significantly upregulated within 15, 45, and 80 days (P < 0.05), but the AST activity was significantly upregulated within 15 and 45 days (P < 0.05), and there was no significant difference within 80 days (P > 0.05). There was no significant difference in the ALT activity between the Control group and the FB5 group at 15, 45, and 80 days (P > 0.05), and there was no significant difference in the AKP activity between 15 and 80 days (P > 0.05), but it decreased at 45 days. The ACP content in the FB5 group increased within 15 days (P < 0.05), but there was no significant difference compared with the Control group at 45 and 80 days. However, compared with the FB0 group, the activities of ACP, AKP, ALT, and AST in the FB5 group almost all decreased at 15, 45, and 80 days (except for the ACP activity at 15 days) (P < 0.05).

[0138] (2) In tilapia serum: The production amounts of ACP, AKP, and ALT in the serum were significantly higher than those in the control group on days 15, 45, and 80. The activity of AST was significantly higher than that in the control group on days 15 and 80 (P < 0.05), but decreased on day 45 (P > 0.05). Compared with the Control group, the activities of AKP, ACP, ALT, and AST in the FB1 group were significantly increased within 15 days. Meanwhile, the activity of ACP in the FB1 group was significantly increased within 45 days (P < 0.05), and the activities of ALT and AST were increased (P > 0.05). Finally, regarding AKP, there was no significant difference between the control group and the FB1 group on day 45. On day 80, compared with the Control group, a significant increase in the activities of AKP and ACP was observed (P < 0.05), a significant decrease in the activity of AST was observed (P < 0.05), and there was no significant difference in the ACP activity between the FB1 groups (P > 0.05). Compared with the Control group, there was no significant difference in the AKP activity in the FB5 group (P > 0.05). The production of ACP, ALT, and AST was significantly upregulated within 15 days, and there was no significant difference in the activities of AKP, ALT, and AST, but the activities of AKP and AST were upregulated (P > 0.05). Compared with the Control group, there was no significant difference in the activities of AKP, ALT, and AST in the 45-day group, but the activities of AKP and AST were upregulated (P > 0.05), and the production of ACP was significantly upregulated (P < 0.05). There was no significant difference in the activities of AKP, ALT, and AST in the 80-day group, but the activities of ALT and AST were downregulated (P > 0.05), and the production of ACP was significantly upregulated (P < 0.05). However, compared with the FB0 group, the activities of ACP, AKP, ALT, and AST in the FB5 group were significantly reduced by approximately 2.0-fold within 15, 45, and 80 days (P < 0.05).

[0139] 3.3. The non-specific detection results of tilapia serum and liver are shown in Table 6:

[0140] Table 6 Non-specific immunofluorescence quantitative analysis of tilapia liver and whole blood

[0141]

[0142]

[0143] As can be seen from Table 6:

[0144] (1) The expression level of IL-1β in the FB0 group increased significantly at 15 d and 80 d, but decreased significantly at 45 d. Compared with the Control group, the expression levels of IL-1β in the FB1 group and FB5 group were down-regulated at 15 d and 45 d, but increased significantly in the FB0 group at 45 d. The hepatic expression level of IL-1β reached the peak at 80 d, but showed a downward trend among the experimental groups. At 80 d, the hepatic expression level of TNF-α reached the peak, and there was still an obvious upward trend among the experimental groups (P < 0.05). Subsequently, its expression showed a downward trend, and the FB5 group decreased significantly by 31.9% compared with the FB0 group (P < 0.05). In the FB1 group and FB5 group, the expression of IFN-γ increased significantly at 45 d (1.2-fold and 8.0-fold) and 80 d (3.5-fold and 2.9-fold). The expression level of Hsp70 in the FB5 group increased significantly by 20.9-fold within 80 d.

[0145] (2) Study the expression of immune-related genes in whole blood: The expression level of IL-1β in whole blood began to change at 15 d, and the expression level continued to increase between the FB0 group and the FB1 group, reaching the peak at 80 d, but there was still a significant decrease in the FB5 group compared with the Control group (P < 0.05); compared with the control group, the expression of CCL3 in the FB0 group and FB1 group was up-regulated, but down-regulated in the FB5 group. Compared with the Control group, the expression of TNF-α in the FB0 group increased significantly at 15 d (1.8-fold) and 80 d (1.4-fold), and decreased at 45 d (P < 0.05). The expression of TNF-α in the FB1 group was up-regulated at 15 d and significantly lower than that of the control group at 45 d and 80 d. The expression of TNF-α in the FB5 group was still significantly decreased at 15, 45, and 80 d (P < 0.05). Compared with the Control group, the expression of MT in the FB0 group and FB1 group was still significantly decreased at 15 d and 45 d (P < 0.05), but showed a downward trend at 80 d (P > 0.05). The expression level of IFN-γ had no obvious effect in the FB0 group (P > 0.05), but was up-regulated in the FB1 group and FB5 group at 15 d (P > 0.05). Compared with the Control group, the expression of IFN-γ in the experimental groups was down-regulated at 45 d and 80 d (P < 0.05). Compared with the Control group, the expression of Hsp70 in the experimental groups was significantly up-regulated at 15 d (P < 0.05). Compared with the Control group, the expression of Hsp70 in the experimental groups was significantly down-regulated at 45 d (P < 0.05). After that, the expression level of Hsp70 in the FB0 group was not significantly affected, but at 80 d, the expression levels of Hsp70 in the FB1 group and FB5 group were up-regulated by 1.1-fold and 1.6-fold of the control group respectively.

[0146] 3.4. The observation results of the microstructure of tilapia liver are as Figure 4 shown:

[0147] Figure 4 It is a histological structure diagram of the liver of tilapia fed with germinated broad bean feed for different days. From Figure 4 it can be seen that the liver of tilapia in the Control group was reddish-brown, with a sharp edge, and the liver surface was smooth and elastic. The livers of tilapia in the FB0 group and the FB1 group were significantly enlarged and fragile, pale in color, softer in texture, with a round and blunt edge, similar to big white liver. At 15 d, the histological morphology of the liver tissue in the Control group was normal, the cell nucleus was located in the center of the cell, there was no infiltration of inflammatory cells, and the number of cell nuclei was the largest. In the FB0 group and the FB1 group, fatty degeneration occurred in the hepatocytes, and a large number of lipid droplet vacuoles of different sizes appeared in the cytoplasm. The histological morphology of the liver tissue in the FB5 group was relatively normal, with a small number of lipid droplet vacuoles. As the feeding time increased, at 45 and 80 d of feeding, the damage to hepatocytes in the FB0 group and the FB1 group was aggravated, some hepatocytes were enlarged, the cell nucleus adhered to the wall or disappeared, and the hepatocytes showed a fishing net-like shape with patchy necrosis areas. However, in the FB5 group, the number of lipid droplet vacuoles decreased and the number of cell nuclei increased. It shows that germinated broad beans can reduce liver damage.

[0148] The above results show that: the germinated broad bean feed provided by the present invention improves the antioxidant capacity of the tilapia body, reduces liver function damage, and reduces inflammatory reactions.

[0149] Experimental Example 4. Effects of germinated broad bean feed on the intestinal health of tilapia

[0150] 1. Experimental method:

[0151] In order to explore the effects of adding germinated broad beans with different days to the feed on the intestinal health of tilapia, the specific grouping is shown in Example 5. The breeding cycle was 80 days. Samples were taken at 15 d, 45 d, and 80 d, and the expression of genes related to intestinal non-specific immunity of tilapia in each group was detected, and the intestinal microscopic structure was observed. The genes related to intestinal non-specific immunity were IL-1β, CCL3, TNF-α, IFN-γ, MT, Hsp70. The detection method was referred to the method of "1.2. Determination of liver non-specific indicators" in Experimental Example 3 for detection. The intestinal microscopic structure was stained with hematoxylin-eosin dye for the sections, and the histological structure of the intestine was observed under an optical microscope.

[0152] 2. Experimental results:

[0153] 2.1. The results of the expression of genes related to intestinal non-specific immunity are as Figure 5 shown:

[0154] Figure 5 It is a diagram of the results of the expression of genes related to intestinal non-specific immunity of tilapia fed with germinated broad bean feed for different days. From Figure 5It can be seen that the expression levels of intestinal IL-1β in the FB1 group and the FB5 group were significantly higher than those in the Control group and the FB0 group (P<0.05), and the FB0 group was higher than the Control group, but there was no significant difference (P>0.05). The expression levels of CCL3 in the FB1 group and the FB5 group were slightly higher than those in the Control group, and the FB0 group was lower than the Control group, with no significant difference (P>0.05). The expression levels of intestinal TNF-α in the FB0 group, the FB1 group, and the FB5 group were all significantly higher than those in the Control group (P<0.05); in the intestine of the FB0 group, Hsp70 and IFN-γ were significantly higher than those in the Control group, and the FB1 group and the FB5 group were significantly lower than the Control group (P<0.05); the intestinal expression level of MT in the experimental group was significantly lower than that in the Control group (P<0.05), and among them, MT in the intestine of the FB0 group and the FB5 group was significantly higher than that in the FB1 group (P<0.05). There was no significant difference in the expression levels of intestinal IL-1β, IFN-γ, and Hsp70 between the FB1 group and the FB5 group.

[0155] 2.2. Results of intestinal microstructure are as Figure 6 shown:

[0156] Figure 6 Microscopic structure observation diagrams of the intestines of tilapia fed with germinated broad bean feed for different days, where: Figure 6 -a is the Control group, Figure 6 -b is the FB0 group, Figure 6 -c is the FB1 group, Figure 6 -d is the FB5 group.

[0157] It can be seen from Figure 6 that: In the intestinal tissue of the Control group, a small amount of intestinal villus epithelium shedding was visible, and many goblet cells were scattered on the mucosal surface; the intestinal villi were long, with a rich number, showing an intertwined branched and finger-like shape, arranged regularly, and there were no intestinal glands; the muscle layer was evenly stained, and the morphological structure of the muscle fibers was normal; no obvious inflammatory reaction was seen ( Figure 6 -a). In the intestinal tissue of the FB0 group, occasional mucosal epithelium shedding was seen, and many goblet cells were scattered on the mucosal surface; the intestinal villi were short and small, mostly showing a short finger-like, conical, and dentate shape, and extensive lamina propria edema was visible, the connective tissue was arranged loosely, a large number of blood vessels were dilated, accompanied by punctate lymphocyte infiltration ( Figure 6 -b). In the intestinal tissue of the FB1 group, more intestinal villus epithelium shedding was visible, and autolysis occurred at the top of some lamina propria. A small number of goblet cells were scattered on the mucosal surface; the intestinal villi were of different lengths, with a rich number, mostly showing a finger-like, conical, and dentate shape, and the local arrangement was irregular, and there were no intestinal glands; the muscle layer was evenly stained, and the morphological structure of the muscle fibers was normal; no obvious inflammatory reaction was seen ( Figure 6 -c). In the intestinal tissue of the FB5 group, more intestinal villus epithelium shedding was visible ( Figure 6-d), a small number of goblet cells can be seen scattered on the mucosal surface; the intestinal villi are thick and abundant, most of which are finger-shaped, conical and dentate, arranged regularly, without intestinal glands; the muscular layer is evenly stained and the morphological structure of muscle fibers is normal; no obvious inflammatory reaction is seen. It can be seen that germinated broad beans can reduce intestinal inflammation.

[0158] The above results show that: the germinated broad bean feed provided by the present invention helps to improve the intestinal tissue structure of tilapia and reduce intestinal inflammation.

[0159] Experimental Example Five, Effect of Deoxynivalenol on Growth Performance of Tilapia

[0160] 1. Experimental method:

[0161] In order to explore the effect of deoxynivalenol on the growth performance of tilapia, 0, 1, and 3 mg / kg of deoxynivalenol (DON) were added to the germinated broad bean feed prepared in Example 2 and Example 4. The breeding period was 80 days. Samples were taken at 15d, 45d, and 80d to measure the weight gain rate of tilapia.

[0162] 3. Experimental results:

[0163] The experimental results are shown in Table 7.

[0164] Table 7 Effect of Deoxynivalenol on Weight Gain Rate of Tilapia

[0165]

[0166] Deoxynivalenol (DON) is one of the most common mycotoxins contaminating feed, with a wide range of toxic effects, such as reduced appetite, weight loss, and metabolic disorders, which are the main pathological results in fish. When the DON concentration in the feed is 1 mg / kg and 3 mg / kg, it can reduce the weight gain rate of tilapia and have an adverse effect on the growth performance of tilapia. As can be seen from Table 7, compared with Example 2, feeding the germinated broad bean feed prepared in Example 4 can effectively alleviate the harmful effects of DON on tilapia and can effectively improve the resistance of tilapia to DON.

[0167] In addition, refer to the method of GB 2761—2017 "National Food Safety Standard Limits of Mycotoxins in Foods" to determine the content of deoxynivalenol in the germinated broad bean feed prepared in Example 2 and Example 4. Among them: the content of deoxynivalenol in the germinated broad bean feed prepared in Example 2 is lower than 800 μg / kg, while the content of deoxynivalenol in the germinated broad bean feed prepared in Example 4 is lower than 200 μg / kg.

[0168] The above embodiments are only illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A germinated broad bean feed for improving the intestinal health of tilapia, characterized in that, It comprises the following components and their parts by weight: Fish meal: 1 - 3 parts, soybean meal: 10 - 20 parts, cottonseed meal: 5 - 20 parts, rapeseed meal: 5 - 20 parts, wheat bran: 8 - 12 parts, rice bran: 8 - 12 parts, wheat middlings: 10 - 25 parts, soybean oil: 1 - 2 parts, choline chloride: 0.4 - 0.6 parts, vitamin premix: 0.2 - 0.4 parts, mineral element premix: 0.2 - 0.5 parts, calcium dihydrogen phosphate: 1 - 3 parts, germinated broad beans: 45 - 55 parts, and mold inhibitor: 0.1 - 0.2 parts.

2. The germinated broad bean feed for improving the intestinal health of tilapia according to claim 1, characterized in that, It consists of the following components and their parts by weight: Fish meal: 1 part, soybean meal: 10 parts, cottonseed meal: 6 parts, rapeseed meal: 8 parts, wheat bran: 10 parts, rice bran: 10 parts, wheat middlings: 10.85 parts, soybean oil: 1 part, choline chloride: 0.5 parts, vitamin premix: 0.25 parts, mineral element premix: 0.3 parts, calcium dihydrogen phosphate: 1.5 parts, germinated broad beans: 50 parts, and mold inhibitor: 0.15 parts, and the mold inhibitor is montmorillonite.

3. The germinated broad bean feed for improving the intestinal health of tilapia according to claim 1 or 2, characterized in that, The germinated broad beans are the germinated broad beans on the 1st - 5th day after the broad beans are soaked and germinated.

4. The germinated broad bean feed for improving the intestinal health of tilapia according to claim 1, wherein The mold inhibitor is modified montmorillonite.

5. The germinated broad bean feed for improving the intestinal health of tilapia according to claim 4, wherein, The preparation method of the modified montmorillonite is as follows: Step A: Add calcium - based montmorillonite into distilled water, hydrate for 1 - 2 h to obtain a suspension; then add sodium carbonate into the suspension, react at a temperature of 50 - 70 °C for 1 - 3 h, dry, and crush to 1000 - 1500 mesh to obtain modified sodium - based montmorillonite; Step B: Add arginine and the modified sodium - based montmorillonite obtained in Step A into distilled water, stir and react for 1 - 3 h, centrifuge, wash the precipitate until neutral, and vacuum - dry to obtain it.

6. The germinated broad bean feed for improving the intestinal health of tilapia according to claim 5, characterized in that, In Step A, the material - liquid ratio of calcium - based montmorillonite to distilled water is 1 g:(8 - 10) mL.

7. The germinated broad bean feed for improving the intestinal health of tilapia according to claim 5, wherein In Step A, the mass concentration of sodium carbonate in the suspension is 4 - 5%.

8. The germinated broad bean feed for improving the intestinal health of tilapia according to claim 5, characterized in that, In Step B, the mass ratio of arginine to modified sodium - based montmorillonite is (5 - 7):

10.

9. The germinated broad bean feed for improving the intestinal health of tilapia according to claim 5, characterized in that, In Step B, the material - liquid ratio of modified sodium - based montmorillonite to distilled water is 1 g:(6 - 8) mL.

10. The preparation method of the germinated broad bean feed for improving the intestinal health of tilapia according to any one of claims 1 to 9, characterized in that, It includes the following steps: Step S1: Mix fish meal, soybean meal, cottonseed meal, rapeseed meal, wheat bran, rice bran, wheat middlings, choline chloride, vitamin premix, mineral element premix, calcium dihydrogen phosphate, germinated broad beans, and mold inhibitor evenly, crush, and pass through a 40 - mesh sieve to obtain mixture I; Step S2: Add soybean oil and distilled water into the mixture I obtained in Step S1, the added amount of distilled water is 25 - 30% of the mass of mixture I, stir evenly, make into sinking pellets with a particle size of 2 mm, and dry at a temperature of 60 - 70 °C to obtain it.

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